Inhibition of DNA polymerases by uracil-DNA glycosylase-cleavable oligonucleotide ligands

ABSTRACT

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising modifying the aptamer by uracil-DNA glycosylase enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. In some aspects, the oligonucleotide aptamers are circular and comprise one or more deoxyuridine nucleotides providing for aptamer-specific recognition and modification of the circular aptamer by the uracil-DNA glycosylase enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing uracil-DNA glycosylase enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one uracil-DNA glycosylase enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific modification of the aptamer by the uracil-DNA glycosylase enzymatic activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.16/210,643, filed Dec. 5, 2018, issued as U.S. Pat. No. 10,724,017,which claims the benefit of U.S. Provisional Patent Application No.62/595,547, filed Dec. 6, 2017, the disclosures of which are hereinincorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “0102384-003US1 SequenceListing.txt,” which was created on Dec. 5, 2017, and is 7 KB in size,are hereby incorporated by reference in their entirety.

BACKGROUND

Aspects of the present invention relate generally to improved methods ofblocking DNA polymerase activity with oligonucleotide aptamers at lowreaction temperatures, and restoring the enzyme activity upon raisingthe reaction temperature (e.g., hot-start methods).

DNA polymerases are enzymes used for synthesis of DNA strands by primerextension, wherein the polymerase-catalyzed DNA synthesis may beinitiated by oligonucleotide primers hybridized to a complementarytemplate DNA. Initiating DNA synthesis from this template-hybridizedprimer, DNA polymerases create complementary DNA strands in the presenceof corresponding nucleotide 5′-triphosphates. Sequence specificity ofnucleotide polymerization, when the oligonucleotide primers bindexclusively to the desired sites and nowhere else, is an importantrequirement in many applications wherein DNA synthesis is used. However,the efficiency and fidelity of DNA synthesis can be reduced when primershybridize to non-complementary DNAs, leading to synthesis of incorrectDNA sequences.

Many so-called ‘Hot Start’ methods have been developed to avoidincorrect primer extension products (e.g., see Paul, N., et al. (2010),for review). One of the most common techniques is based on use ofoligonucleotide aptamers (Jayasena S. D., 1999). Aptamers offer a numberof advantages over other reported methods. Using a method of molecularevolution (SELEX), they can be quickly engineered in a test tube andthen readily and inexpensively manufactured by chemical synthesis.Ideally, an aptamer should: (i) completely block DNA polymerase at lowtemperatures, and (ii) provide no blockage effect at the desiredelevated reaction temperature. Unfortunately, this is very difficult, ifnot impossible to achieve, and the aptamer structure usually representsa compromise between these two key requirements. New methods, therefore,are needed to improve control of aptamer activity in reaction mixturescontaining DNA polymerases.

Particular aspects provide methods for activating an aptamer-inactivatedDNA polymerase, comprising: providing a reaction mixture suitable forDNA synthesis, the reaction mixture comprising (i) a DNA polymerase,(ii) a uracil-DNA glycosylase enzymatic activity, and (iii) a DNApolymerase-binding oligonucleotide aptamer that comprises a hairpinstructure having a stem sequence portion and a loop sequence portion,wherein the loop sequence portion comprises one or more deoxyuridinenucleotide(s) modifiable by the uracil-DNA glycosylase enzymaticactivity, and the aptamer is present in an amount sufficient to inhibitDNA synthesis activity of the DNA polymerase in the reaction mixture;and modifying the aptamer by the uracil-DNA glycosylase enzymaticactivity to form a modified aptamer having less or no inhibitory effecton the DNA polymerase, thereby activating or enhancing the DNA synthesisactivity of the DNA polymerase, to start and/or increase DNA synthesisin the reaction mixture. In the methods, for example, modifying theaptamer may be facilitated by use of a reaction temperature thatfacilitates the DNA polymerase activity and/or the uracil-DNAglycosylase enzymatic activity. In the methods, modifying the aptamermay be facilitated by increasing the temperature of the reaction mixturefrom a first temperature to a second temperature that activates or morestrongly facilitates the uracil-DNA glycosylase enzymatic activity. Inthe methods, providing the reaction mixture may comprise dissolving adried form of at least one of the (i) DNA polymerase, (ii)oligonucleotide aptamer, and (iii) uracil-DNA glycosylase enzymaticactivity, into an aqueous solution. In the methods, the DNA synthesismay result in DNA amplification in the reaction mixture (e.g., whereinthe DNA amplification is an isothermal amplification reaction, and/orwherein the DNA amplification is PCR). The methods may comprisedetecting a presence of a target DNA in the reaction mixture, and/ormeasuring an amount of a target DNA in the reaction mixture. In themethods, the oligonucleotide aptamer may be circular. In the methods,the uracil-DNA glycosylase enzymatic activity may be effective to modifythe oligonucleotide aptamer by generating at least one abasic sitewithin the loop sequence portion thereof. In the methods, the uracil-DNAglycosylase may be or comprise Afu Uracil-DNA Glycosylase. In themethods, the loop sequence portion may be or comprise a nucleotidesequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) wherein one or more thymidinenucleotides at positions 1, 2, 10, 11, and 12 of the SEQ ID NO:23sequence are substituted by one or more deoxyuridine nucleotides.

Additional aspects provide kits for activating an aptamer-inactivatedDNA polymerase, comprising: a uracil-DNA glycosylase enzymatic activity;and a DNA polymerase-binding oligonucleotide aptamer sequence that iscapable of forming a hairpin structure having a stem sequence portionand a loop sequence portion, wherein the loop sequence portion comprisesone or more deoxyuridine nucleotides modifiable by the uracil-DNAglycosylase enzymatic activity. In the kits, the loop sequence portionmay be or comprise a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ IDNO:23) wherein one or more thymidine nucleotides at positions 1, 2, 10,11, and 12 of the SEQ ID NO:23 sequence are substituted by one or moredeoxyuridine nucleotides. In the kits, the oligonucleotide aptamer maybe a circular molecule. In the kits, the uracil-DNA glycosylaseenzymatic activity may be effective to modify the oligonucleotideaptamer by generating at least one abasic site within the loop sequenceportion. In the kits, the uracil-DNA glycosylase may be or comprise AfuUracil-DNA Glycosylase.

Further aspects provide reaction mixtures for use in a method of DNAsynthesis, which reaction mixture comprises: (i) a DNA polymerase, and(ii) a DNA polymerase-binding oligonucleotide aptamer that comprises ahairpin structure having a stem sequence portion and a loop sequenceportion, wherein the loop sequence portion comprises one or moredeoxyuridine nucleotides modifiable by a uracil-DNA glycosylaseenzymatic activity, and the aptamer is present in an amount sufficientto inhibit DNA synthesis activity of the DNA polymerase in the reactionmixture. The reaction mixtures may further comprise (iii) a uracil-DNAglycosylase enzymatic activity sufficient, under suitable conditions, tomodify the oligonucleotide aptamer to reduce or eliminate binding of theoligonucleotide aptamer to the DNA polymerase, thereby activating orenhancing the DNA synthesis activity of the DNA polymerase. In thereaction mixtures, at least one of the DNA polymerase activity and/orthe uracil-DNA glycosylase enzymatic activity may be temperaturedependent. In the reaction mixtures, the uracil-DNA glycosylaseenzymatic activity may increase with increasing temperature of thereaction mixture from a first temperature to a second temperature thatactivates or more strongly facilitates the uracil-DNA glycosylaseenzymatic activity. In the reaction mixtures, the DNA polymerase, and/oroligonucleotide aptamer, and/or uracil-DNA glycosylase enzymaticactivity may be present in the reaction mixture in a dried state. In thereaction mixtures, the loop sequence portion may be or comprise anucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) wherein one ormore thymidine nucleotides at positions 1, 2, 10, 11, and 12 of the SEQID NO:23 sequence are substituted by one or more deoxyuridinenucleotides. In the reaction mixtures, the oligonucleotide aptamer maybe a circular molecule. In the reaction mixtures, the uracil-DNAglycosylase enzymatic activity may be effective to modify theoligonucleotide aptamer by generating at least one abasic site withinthe loop sequence portion. In the reaction mixtures, the uracil-DNAglycosylase may be or comprise Afu Uracil-DNA Glycosylase. The reactionmixtures may further comprise one or more of dATP, dCTP, dGTP, and/ordTTP, and/or Mg²⁺ ion.

Yet further aspects provide an oligonucleotide aptamer, comprising a DNApolymerase-binding nucleic acid sequence that is capable of forming ahairpin structure having a stem sequence portion and a loop sequenceportion, wherein the loop sequence portion is or comprises a nucleotidesequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23), wherein one or morethymidine nucleotides at positions 1, 2, 10, 11, and 12 of the SEQ IDNO:23 sequence are substituted by one or more deoxyuridine nucleotidesmodifiable by a uracil-DNA glycosylase enzymatic activity (e.g., by AfuUracil-DNA Glycosylase). The oligonucleotide aptamers may be circularmolecules. The circular oligonucleotide aptamers may comprises a duplexstem sequence portion positioned between two loop sequence portions. Inthe circular oligonucleotide aptamers comprising a duplex stem sequenceportion positioned between two loop sequence portions, the two loopsequence portions may be the same or different sequences. Theoligonucleotide aptamers may be in combination with a DNA polymerase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, according to particular exemplary aspects, a portion of ahuman β2-microglobulin gene sequence (SEQ ID NO:4), forward and reverseprimers (SEQ ID NOS:1-2, respectively) and a 22-mer fluorescent probe(SEQ ID NO:3), which were used in exemplary 5′-nuclease PCR assays ofthe present invention from which exemplary results are shown in FIGS. 3and 5. The primers and probe are shown aligned with an amplified humanβ2-microglobulin fragment sequence in 5′→3′ orientation as indicated.

FIG. 2 shows, according to particular exemplary aspects, structures ofnoncircular stem-loop deoxyribonucleotide aptamers used in exemplaryworking Examples of the invention with uracil-DNA glycosylase (FIGS. 3A,3B, and 7A-F). The symbol “U” is deoxyribouridine nucleotide. AptamersSEQ ID NOS:6-12 are the structural analogs of unmodified aptamer SEQ IDNO:5 incorporating deoxyuridine at various positions within the loopsequence.

FIGS. 3A and 3B show, according to particular exemplary aspects, theresults of fluorescence monitoring of reaction mixtures during PCR(real-time curves) in the presence of the individual aptamers listed inFIG. 2. Sequences of the amplified human β2-microglobulin template,primers and 22-mer FRET probe used in these PCR assays are as shown inFIG. 1. Dashed lines are real-time curves obtained in the absence of anyaptamer. Experiments were conducted in the absence (FIG. 3A) or presence(FIG. 3B) of Afu Uracil-DNA Glycosylase. Experimental details areprovided below in “Example 3.”

FIG. 4 shows, according to particular exemplary aspects, structures ofeight additional circular stem-loop deoxyribonucleotide aptamers SEQ IDNOS:13-20 (right column). The central column shows linearoligonucleotide sequences used to prepare the corresponding circularaptamers SEQ ID NOS:13-20 (right column) as described herein in Example2. The symbol “p” indicates a 5′-phosphate moiety. Circular stem-loopdeoxyribonucleotide aptamers SEQ ID NOS:14-20 are structural homologs ofthe unmodified circular aptamer SEQ ID NO:13 representing all possible(seven), single deoxythymidine-to-deoxyuridine substitutions (marked bysymbol “U”) within the loop sequence as indicated. These aptamers wereused in the 5′-nuclease PCR assays of FIGS. 5A and 5B.

FIGS. 5A and 5B show, according to particular exemplary aspects, theresults of fluorescence monitoring of reaction mixtures during PCR(real-time curves) in the presence of the individual aptamers listed inFIG. 4. Sequences of the amplified human β2-microglobulin template,primers and 22-mer FRET probe used in these PCR assays are as shown inFIG. 1. Dashed lines are real-time curves obtained in the absence of anyaptamer. Experiments were conducted in the absence (FIG. 5A) or presence(FIG. 5B) of Afu Uracil-DNA Glycosylase. Experimental details areprovided below in “Example 3.”

FIG. 6 shows, according to particular exemplary aspects, a scheme of areaction used in the working Examples to detect and measure DNApolymerase activity. To enable use over a wide range of temperatures(e.g., up to 70° C.), the depicted hairpin-like fluorescent probe wasdesigned to have a G/C-rich stem in the duplex segment and a 5′- . . .GAA . . . hairpin-stabilizing loop (e.g., see Yoshizawa S., et al.,1994). Extension of this hairpin in a reaction buffer in the presence ofdeoxyribonucleoside 5′-triphosphates (dNTPs) and a DNA polymeraseresults in a fluorescent signal that directly correlates with the DNApolymerase activity in the reaction.

FIGS. 7A through 7F show, according to particular exemplary aspects,uracil-DNA glycosylase-induced activation of Taq (FIG. 7D), Phusion®(FIG. 7B), Q5® (FIG. 7C), Vent® (FIG. 7A), Deep Vent® (FIG. 7E) and Bstlarge fragment (FIG. 7F) DNA polymerases that were initially deactivated(i.e., “inhibited” or “blocked”) by the presence of aptamer SEQ ID NO:12(♦ curves). FIGS. 7A through 7F also show the change of fluorescencewith time in the absence of uracil-DNA glycosylase for theaptamer-blocked (∘) and unblocked (□) DNA polymerase. The DNA polymeraseactivity was monitored by extension of the self-priming hairpin-likefluorescent probe SEQ ID NO:21 (see FIG. 6), which was present in thereaction mixture with all four dNTPs in a magnesium-containing buffer.In all cases, experiments were performed at 60 or 65° C., as indicatedin each Figure. The structure of aptamer SEQ ID NO:12 is shown in FIG.2, and details of the experimental setup, results analysis andconclusions are provided below in “Example 4.”

DETAILED DESCRIPTION Definitions

Terms and symbols of biochemistry, nucleic acid chemistry, molecularbiology and molecular genetics used herein follow those of standardtreatises and texts in the field (e.g., Sambrook, J., et al., 1989;Kornberg, A. and Baker, T., 1992; Gait, M. J., ed., 1984; Lehninger, A.L., 1975; Eckstein, F., ed., 1991, and the like). To facilitateunderstanding of particular exemplary aspects of the invention, a numberof terms are discussed below.

In particular aspects, “aptamer” or “oligonucleotide aptamer” refersherein to an oligonucleotide that can form a secondary structure such ashairpin or stem-loop structure that is capable of binding to a DNApolymerase and blocking its DNA synthesis enzymatic activity. Examplesof such aptamers and methods of their sequence selection (design) can befound, for instance, in Yakimovich, O. Yu., et al. (2003); Jayasena, S.D. (1999); U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D.,which are incorporated here by reference. The phrase “aptamer, thatbinds to the DNA polymerase, in an amount sufficient to inhibit DNAsynthesis activity of the DNA polymerase,” as used herein, means thatthe DNA synthesis activity of the DNA polymerase is at least partiallyinhibited (e.g., inhibited to a level in the range of from about 1% toabout 99.990%). Any level of aptamer inhibition of the DNA synthesisactivity of the DNA polymerase can provide an advantage for DNAsynthesis, and thus according to particular preferred hot start aspectsof the present invention, the DNA synthesis activity of the DNApolymerase is substantially inhibited (e.g., inhibited to a level in therange of about 80% to 99.99%, or to any subrange or level therein), orcompletely (100%) inhibited, providing an advantage over other ‘hotstart’ technologies (e.g., Paul N. et al, 2010). Likewise, in thedisclosed methods, “modifying the aptamer by a uracil-DNA glycosylaseenzymatic activity to reduce or eliminate the binding of theoligonucleotide aptamer to the DNA polymerase and activate the DNAsynthesis activity of the DNA polymerase” is preferably complete (100%)or substantially complete (e.g., inhibited to a level in the range ofabout 800% to 99.990%, or to any subrange or level therein), but can bepartial (e.g., inhibited to a level in the range of from about 1% toabout 99.99%), as exemplified herein (e.g., FIGS. 3, 5, and 7).

An oligonucleotide aptamer may comprise ribo- or 2′-deoxyribonucleotidesor a combination thereof. Oligonucleotide aptamers may be modified.Regarding the aptamers, the term “modification” is used herein in twodifferent aspects, wherein the aptamers can be (i) modifiedsynthetically, e.g., during the oligonucleotide synthesis, and (ii)enzymatically-modified in the context of or during DNA synthesisreactions. Synthetically, the aptamers may incorporate any kind and/ornumber of structural modifications across the length of the aptamer(e.g., in the middle or at the ends of the oligonucleotide chain). Theterm “structural modifications” refers to any chemical substances suchas atoms, moieties, residues, polymers, linkers or nucleotide analogs,etc., which are usually of a synthetic nature and which are not commonlypresent in naturally-occurring nucleic acids. As used herein, the term“structural modifications” also include nucleoside or nucleotide analogswhich are rarely present in naturally-occurring nucleic acids includingbut not limited to inosine (hypoxanthine), 5-bromouracil,5-methylcytosine, 5-iodouracil, 2-aminoadenosine, 6-methyladenosine,preudouridine, deoxyuridine, and the like. The structural modificationscan be “duplex-stabilizing modifications.” “Duplex-stabilizingmodifications” refer to structural modifications, the presence of whichin double-stranded nucleic acids provides a duplex-stabilizing effectwhen compared in thermal stability, usually measured as “Tm,” withrespective nucleic acid complexes that have no such structuralmodification and, e.g., comprise natural nucleotides. Duplex-stabilizingmodifications are structural modifications that are most commonlyapplied in synthesis of probes and primers, as represented by modifiednucleotides and ‘tails’ like intercalators and minor groove binders as,for example, disclosed in U.S. Pat. No. 8,349,556 to Kutyavin, I. V.;U.S. Pat. No. 7,794,945 to Hedgpeth, J., et al.; U.S. Pat. No. 6,127,121to Meyer, Jr., R. B., et al.; U.S. Pat. No. 5,801,155 to Kutyavin, I.V., et al., and the references cited in. Duplex-stabilizingmodifications can be used to prepare aptamers of the invention, forexample, to improve thermal stability of stem (duplex) structures ofhairpin-like aptamers. In preferred methods of the invention, theoligonucleotide aptamers are modified in (e.g., during) the DNAsynthesis reactions using enzymatic activity of one or more uracil-DNAglycosylase(s). In this aspect, the terms “modify,” “modification,” and“structural modifications” mean changes in the original syntheticstructure of the aptamers. The change is triggered by a uracil-DNAglycosylase activity, resulting in removal of one or more uracil basesto produce respective abasic site(s). In the methods of the invention,these uracil-DNA glycosylase-triggered structural modifications reduceor eliminate the ability of the oligonucleotide aptamer to bind to theDNA polymerase and block or reduce its activity in the reaction mixture.In particular embodiments, the hairpin-like aptamers incorporatedeoxyuridine nucleotides within the loop sequence thereof. Thehairpin-like aptamers of the invention can be circular molecules.

In particular aspects, the term “secondary structure” refers to anintramolecular complex formation of one sequence in a poly- oroligonucleotide with another sequence in the same polymer due tocomplete or partial complementarity between these two sequences formedbased on the principal rules of Watson-Crick base pairing. The terms“hairpin” structure or “stem-loop” structure as referred to hereindescribe elements of secondary structure, and both terms refer to adouble-helical region (stem) formed by base pairing betweencomplementary sequences within a single strand RNA or DNA.

As used herein, the term “uracil-DNA glycosylases” refer to enzymes thatremove uracil bases or uracil base analogs while leaving thesugar-phosphate backbone intact, creating an apurinic/apyrimidinic site,commonly referred herein to as an “abasic site.” The term “DNApolymerase” refers to an enzyme that catalyzes synthesis of deoxyribonucleic acids (DNAs), most commonly double-stranded DNAs, usingsingle-stranded DNAs as “templates.” The DNA synthesis is usuallyinitiated by an oligonucleotide primer that is hybridized to a templatestrand. Starting from this template-hybridized primer, DNA polymerasecreates a Watson-Crick complementary strand in the presence of2′-deoxyribonucleotide 5′-triphosphates (dNTPs). The term “DNApolymerase,” as used herein, also incorporates “reverse transcriptases,”enzymes which can perform DNA synthesis using single-strandedribonucleic acids (RNAs) as template strands.

“Polynucleotide” and “oligonucleotide” are used herein interchangeablyand in each case means a linear polymer of nucleotide monomers.Polynucleotides typically range in size from a few monomeric units,e.g., 5-60, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. The exact size will depend on manyfactors, which in turn depends on the ultimate function or use of theoligonucleotide. The oligonucleotides may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription, ora combination thereof. Unless otherwise specified, whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters, for example, 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23), it isunderstood herein, unless otherwise specified in the text, that thenucleotides are in 5′→3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,and “T” denotes deoxythymidine. Usually DNA polynucleotides comprisethese four deoxyribonucleosides linked by phosphodiester linkage whereasRNA comprises uridine (“U”) in place of “T” for the ribose counterparts.

The terms “oligonucleotide primer” and/or “primer” refer to asingle-stranded DNA or RNA molecule that hybridizes to a target nucleicacid and serves to prime enzymatic synthesis of a second nucleic acidstrand in the presence of a DNA polymerase. In this case, the targetnucleic acid “serves as a template” for the oligonucleotide primer.

In particular aspects, the terms “complementary” or “complementarity”are used herein in reference to the polynucleotide base-pairing rules.Double-stranded DNA, for example, consists of base pairs wherein, forexample, G complexes or pairs with C via formation of a three hydrogenbond complex, and A complexes or pairs with T via formation of a twohydrogen bond complex, such that G is regarded as being complementary toC, and A is regarded as being complementary to T. In this sense, forexample, an oligonucleotide 5′-GATTTC-3′ is complementary to thesequence 3′-CTAAAG-5′ via intrastrand G:C and A:T hydrogen bondinginteractions. Complementarity may be “partial” or “complete.” In partialcomplementarity, only some of the nucleic acids bases are matchedaccording to the base pairing rules. The terms may also be used inreference to individual nucleotides and oligonucleotide sequences withinthe context of polynucleotides (e.g., inter-strand complementarity). Theterms “complementary” or “complementarity” refer to the most common typeof complementarity in nucleic acids, namely Watson-Crick base pairing asdescribed above, although the oligonucleotides may alternatelyparticipate in other types of “non-canonical” pairings like Hoogsteen,wobble and G-T mismatch pairing.

The terms “natural nucleosides,” refers to the art-recognized four2′-deoxyribonucleosides (usually named herein as “deoxynucleosides” or“deoxyribonucleosides”) that are found in DNAs isolated from naturalsources. Natural nucleosides are deoxyadenosine, deoxycytidine,deoxyguanosine, and deoxythymidine. The term also encompasses theirribose counterparts, with uridine (U) in place of thymidine. The samename variations are applied herein to “natural nucleotides.”

As used herein, the terms “unnatural nucleosides” or “modifiednucleosides” refer to nucleoside analogs that are different in theirstructure from those natural nucleosides for DNA and RNA polymers. Somenaturally occurring nucleic acids contain nucleosides that arestructurally different from the natural nucleosides defined above, forexample, DNAs of eukaryotes may incorporate 5-methyl-cytosine, and tRNAscontain many nucleoside analogs. However, as used herein, the terms“unnatural nucleosides” or “modified nucleosides” encompasses thesenucleoside modifications even though they can be found in naturalsources. For example, ribothymidine as well as deoxyuridine are examplesof unnatural nucleotides referred to herein.

The term “reaction mixture” generally means herein a solution containingall the necessary reactants for performing DNA synthesis such as a DNApolymerase, oligonucleotide primer(s), template polynucleotide,deoxyribonucleoside 5′-triphosphates, reaction cofactors (e.g.,magnesium or manganese ions), etc. The reaction mixture can incorporateother reaction components that help to improve the DNA synthesis (e.g.,buffering and salt components, detergents, proteins like bovine serumalbumin (BSA), scavengers, etc.) or components that are necessary fordetection of the newly synthesized DNA molecules such as, for example,fluorescent dyes and oligonucleotide probes. A reaction mixture isusually prepared at low temperatures at which enzymatic components areinactive, for example, by mixing the components on ice at ˜0° C. Whenthe reactions are ready, the mixtures can be heated to the desiredreaction temperatures. In this aspect, the term “reaction temperature”refers to a temperature or a temperature range at which DNA synthesis isperformed. In case of PCR reactions, it is usually taken as the lowestthermo-cycling temperature, commonly called the annealing temperature.

The symbol “dNTPs” is an abbreviation of a mixture of all four naturaldeoxynucleoside 5′-triphosphates that are useful to facilitate primerextension with a DNA polymerase and/or amplification. Respectively, theabbreviations “dATP,” “dCTP,” “dGTP,” and “dTTP” correspond to theindividual nucleotides. In some embodiments, the four dNTPs are presentat equal concentrations. In other embodiments, the concentrations of thedNTPs are not all identical. In some embodiments, fewer than all fourdNTPs are present. For example, only one dNTP may be present, or apair-wise combination, or three of four dNTPs may be present in themixture.

In some aspects, “amplification” and “amplifying” deoxyribonucleicacids, in general, refer to a procedure wherein multiple copies of DNAof interest are generated. The DNA amplification can be performed at aconstant temperature using “isothermal amplification reactions.”Examples of isothermal amplification reactions include, but are notlimited to, Strand Displacement Amplification (SDA) (U.S. Pat. No.5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 toDattagupta, N., et al.), Rolling Circle amplification (RCA) (U.S. Pat.No. 5,854,033 to Lizardi, P.), Loop-Mediated Amplification (LMA) (U.S.Pat. No. 6,410,278 to Notomi, T. and Hase, T.), isothermal amplificationusing chimeric or composite RNA/DNA primers (U.S. Pat. No. 5,824,517 toCleuziat, P. and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kurn, N.),Nucleic Acid Sequence-Based Amplification (NASBA) (U.S. Pat. No.6,063,603 to Davey, C. and Malek, L. T.), and many other methods.

“PCR” is an abbreviation of “polymerase chain reaction,” anart-recognized nucleic acid amplification technology (e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). Commonly used PCRprotocol employs two oligonucleotide primers, one for each strand,designed such that extension of one primer provides a template for theother primer in the next PCR cycle. Generally, a PCR reaction consistsof repetitions (cycles) of (i) a denaturation step that separates thestrands of a double-stranded nucleic acid, followed by (ii) an annealingstep, which allows primers to anneal to positions flanking a sequence ofinterest on a separated strand, and then (iii) an extension step thatextends the primers in a 5′ to 3′ direction, thereby forming a nucleicacid fragment complementary to the target sequence. Each of the abovesteps may be conducted at a different temperature using an automatedthermocycler. The PCR cycles can be repeated as often as desiredresulting in an exponential accumulation of a target DNA fragment whosetermini are usually defined by the 5′-ends of the primers used. Althoughconditions of PCR can vary in a broad range, a double-stranded targetnucleic acid is usually denatured at a temperature of >90° C., primersare annealed at a temperature in the range of about 50-75° C., and theextension is preferably performed in a 72-75° C. temperature range. InPCR methods, the annealing and extension can be combined into one stage(i.e., using a single temperature). The term “PCR” encompasses itsnumerous derivatives such as “RT-PCR,” “real-time PCR,” “nested PCR,”“quantitative PCR,” “multiplexed PCR,” “asymmetric PCR,” and the like.

“Real-time detection” means an amplification reaction for which theamount of reaction product, i.e., target nucleic acid, is monitored asthe reaction proceeds. Real-time detection is possible when alldetection components are available during the amplification and thereaction composition and conditions support both stages of the reaction;the amplification and the detection.

As used herein, the term “kit” refers to any system for deliveringmaterials. In the context of reaction assays, such delivery systemsinclude elements allowing the storage, transport, or delivery ofreaction components such as oligonucleotides, buffering components,additives, reaction enhancers, enzymes, and the like in the appropriatecontainers from one location to another commonly provided with writteninstructions for performing the assay. Kits may include one or moreenclosures or boxes containing the relevant reaction reagents andsupporting materials. The kit may comprise two or more separatecontainers wherein each of those containers includes a portion of thetotal kit components. The containers may be delivered to the intendedrecipient together or separately.

In general, the term “design” in the context of the methods has broadmeaning and in certain respects is equivalent to the term “selection.”For example, the terms “primer design” and “aptamer design” can mean orencompass selection of a particular oligonucleotide structure includingthe nucleotide primary sequence and structural modifications (e.g.,labels, modified nucleotides, linkers, etc.). In particular aspects, theterms “system design” and “assay design” relate to the selection of any,sometimes not necessarily to a particular, methods including allreaction conditions (e.g., temperature, salt, pH, enzymes, including theaptamer-modifying enzymes and DNA polymerase, oligonucleotide componentconcentrations, etc.), structural parameters (e.g., length and positionof primers and probes, design of specialty sequences, etc.), and assayderivative forms (e.g., post-amplification, real time, immobilized, FRETdetection schemes, etc.) chosen to amplify and/or to detect the nucleicacids of interest.

Reversible Blocking DNA Synthesis Activity of DNA Polymerases UsingOligonucleotide Stem-Loop Aptamers of the Invention.

Prior art applications of oligonucleotide aptamers during DNA synthesisare directed at blocking DNA polymerase, preferably completely, at lowtemperatures, while releasing the DNA polymerase activity, preferablycompletely, at an elevated reaction temperature. It is difficult,however, to achieve complete ‘block-and-release’ formats usingconventional aptamer-based methods (e.g., Yakimovich, O. Yu., et al.(2003); Jayasena, S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L. andJayasena, S. D. (1997)). Effective blockage of DNA polymerase at lowtemperatures commonly leads to ineffective release of the enzyme at theelevated reaction temperature and vice versa. Aspects of the presentinvention provide a solution to this long-standing problem in the art.As in the conventional approaches cited above, the DNA polymeraseactivity is blocked or reduced in methods of the invention by thepresence of an oligonucleotide hairpin-like aptamer that binds to theDNA polymerase, blocking the DNA synthesis activity of the DNApolymerase. Unlike prior art techniques, however, in methods of theinvention, the aptamer-inactivated DNA polymerase is activated byproviding, to a DNA synthesis reaction mixture, one or more uracil-DNAglycosylases that recognize the oligonucleotide aptamer as a substrateand modify its structure. This structural modification reduces oreliminates the binding of the oligonucleotide aptamer to the DNApolymerase and thereby reactivates the DNA synthesis activity of the DNApolymerase.

In some embodiments of the invention, activation of anaptamer-inactivated DNA polymerase in a reaction mixture, comprising (i)a DNA polymerase, (ii) oligonucleotide aptamer in an amount effective toinhibit the DNA synthesis activity of the DNA polymerase, (iii)uracil-DNA glycosylase(s) and other components necessary for DNAsynthesis, is facilitated using a reaction temperature that accelerates(or facilitates) both DNA polymerase and aptamer-modifying enzymeactivities. For example, the reaction mixture can be prepared at a lowtemperature (first temperature) at which a DNA polymerase is effectivelyblocked by an aptamer and a uracil-DNA glycosylase enzyme hassufficiently reduced or preferably no activity (e.g., at 0° C.), andthen the activation of the aptamer-inactivated DNA polymerase isfacilitated by heating the reaction to a temperature (secondtemperature) that accelerates or facilitates the uracil-DNA glycosylaseenzymatic activity. If necessary, a DNA polymerase can be activated bythe uracil-DNA glycosylase(s) at any temperature below the reactiontemperature for DNA synthesis. This approach can be applied, forexample, when a particular uracil-DNA glycosylase enzyme is unstable atthe reaction temperature for DNA synthesis, for example, due todenaturation. In this case, the DNA polymerase is first activated at anintermediate temperature wherein the uracil-DNA glycosylase is activeand then heated to the reaction temperature to perform DNA synthesis.

In some aspects, the reaction mixture is created by addition of aqueoussolution to one or more reaction components which are initially in adried state as disclosed and described, for example, in U.S. Pat. No.3,721,725 to Briggs, A. R. and Maxwell, T. J. (1973) (incorporatedherein by reference). For example, in some methods of the invention forDNA amplification and detection, the aqueous solution can be a samplesolution or solution that contains one or more polynucleotide templatesfor DNA synthesis, whereas all other reaction components, or particulardesired reactions components or desired combinations thereof such as theDNA polymerase, aptamer, uracil-DNA glycosylase enzyme(s), dNTPs,catalytic cofactors like magnesium (Mg2+) or manganese (Mn2+) salt(e.g., chloride salts), buffering components, detergents, proteins likebovine serum albumin (BSA), scavengers, etc., are present in a drystate.

In some aspects, DNA synthesis results in DNA amplification in thereaction mixture. The DNA amplification can be an isothermalamplification reaction, for example, as described in U.S. Pat. No.5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 toDattagupta, N., et al.; U.S. Pat. No. 5,854,033 to Lizardi, P.; U.S.Pat. No. 6,410,278 to Notomi, T. and Hase, T.; U.S. Pat. No. 5,824,517to Cleuziat, P. and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kurn, N.;U.S. Pat. No. 6,063,603 to Davey, C. and Malek, L. T., and many othermethods. In other aspects, the DNA amplification can be a PCR reaction(e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). Inmethods of the invention, the DNA amplification may be performed fordetection and/or for measuring an amount of a target DNA in the reactionmixture.

Aptamers of the invention fold into hairpin secondary structures havingstem and loop sequence portions, wherein the loop sequence portionscontain one or more deoxyuridine nucleotide(s) that can be recognizedand removed from the aptamer nucleotide sequence by a uracil-DNAglycosylase enzymatic activity. Relative to unmodified aptamers, theglycosylase-modified aptamers of the invention have reduced or noinhibitory effect on the DNA polymerase, thereby activating or enhancingthe DNA synthesis activity of the DNA polymerase, to start and/orincrease DNA synthesis in the reaction mixture.

The oligonucleotide aptamers as well as oligonucleotide primers andprobes can be prepared by any method of oligonucleotide synthesisdescribed in the art, but preferred is the most modern chemistry basedon (2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites (Example 1).Respectively protected nucleotides and their numerous derivatives,linkers, dyes, tails, solid supports, and other necessary components canbe prepared by methods of organic chemistry or obtained from marketproviders such as, for example, Glen Research and BiosearchTechnologies. Suppliers such as Integrated DNA Technologies andBiosearch Technologies also offer oligonucleotide custom synthesisincluding numerous structural modifications including deoxyuridinenucleotides. For a particular DNA polymerase in the methods of theinvention, selection of an aptamer structure, including one or moredeoxyuridine nucleotide(s), their location within the loop sequence, anduracil-DNA glycosylase enzymes, is intended to achieve (i) completedeactivation of the DNA polymerase at the initial reaction assemblytemperature, (ii) substantially complete or complete (or as much aspossible) deactivation of the DNA polymerase at the elevated reactiontemperature for DNA synthesis, and (iii) substantially complete orcomplete (or as much as possible) activation of this enzyme at the DNAsynthesis reaction temperature once the aptamer has been modified by theuracil-DNA glycosylase enzymatic activity. Preferably, the prospectiveuracil-DNA glycosylases should not interfere with DNA synthesis or DNAamplification. The location and number of deoxyuridines within anaptamer loop sequence, as well as the rate and efficiency of theuracil-DNA glycosylases is taken into consideration. Preference is givento a number of deoxyuridine and/or their locations within an aptamerloop sequence that have little or no negative effect on stability of theaptamer-polymerase complex, but sufficiently disturb structuralintegrity of the aptamer and its ability to bind to a DNA polymeraseafter modification by a uracil-DNA glycosylase to provide optimal DNApolymerase activation. Use of deoxyuridine nucleotides within loopsegments of noncircular (SEQ ID NOS:6-12) and circular (SEQ IDNOS:14-20) hairpin-like aptamers is illustrated in FIGS. 3 and 5,respectively. Surprisingly, positioning of a single base modification inthe loop segments of both noncircular and circular aptamers showedexcellent results in a number of exemplary assays herein.

According to the prior art (Yakimovich, O. Yu., et al. (2003); Jayasena,S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D.),efficiency of hairpin-type aptamer binding to DNA polymerase isdetermined by (i) a substantially conservative loop segment sequence,and (ii) the length of the stem duplex, which preferably needs to be atleast 17 base pairs long (Yakimovich, O. Yu., et al. (2003). Accordingto particular aspects disclosed herein, the sequence and the structureof the stem fragment is another important factor affecting the stabilityof an aptamer-polymerase complex. For example, the 17-base pair longstem sequence of noncircular aptamer SEQ ID NO:5 used in the presentworking Examples was empirically determined, and comprises a A-T richduplex surrounded by G-C clamps (see FIG. 2). This aptamer and many ofits derivatives (SEQ ID NOS:6-12) were nonetheless effective in blockingnot only Taq polymerase, but also many other DNA polymerases (FIGS.7A-7F). As exemplified herein (FIGS. 5A and 5B), circularization ofaptamers (Examples 2 and 3) allowed (i) reduction of the hairpin stemlength by 3 base pairs and (ii) use of a relatively unstable A-T-richsequence in the stem design, while maintaining utility of the aptamersin methods of the invention. Regardless of these changes in the stemdesign parameters, generally regarded in the prior art asnegative/unfavorable changes, unmodified circular aptamer SEQ ID NO:13(FIG. 4) as well as many of its deoxyuridine-modified derivatives (SEQID NOS:14-20) inactivated Taq DNA polymerase in PCR assays (FIG. 5A) aseffectively as unmodified noncircular aptamer SEQ ID NO:5 (FIG. 2) andits deoxyuridine derivatives SEQ ID NOS:6-12 (FIG. 3A). Circularizationof the aptamers leads to formation of second loop sequence in additionto the conserved loop sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) used inthe examples provided herein. This second loop sequence can be of anylength, sequence and sequence composition including the same conservedsequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23), and including wherein thesecond loop sequence portion comprises a nucleotide sequence5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) wherein one or more thymidinenucleotides at positions 1, 2, 10, 11, and 12 of the SEQ ID NO:23sequence are substituted by one or more deoxyuridine nucleotides.

Comparison of the results shown in FIGS. 7B and 7D points to yet anothersurprising result. First, regardless of the difference in reactiontemperature, the same aptamer (SEQ ID NO:12) more efficiently blockedPhusion polymerase at 65° C. than Taq polymerase at 60° C. According toadditional surprising aspects of the invention, therefore, the optimalblocking sequence of an aptamer hairpin duplex may bepolymerase-specific. Second, the randomly-chosen duplex sequence used inaptamers SEQ ID NOS:5-12 may not be the most optimal one, and it may befurther optimized by base pair changes for even better polymeraseblockage. Third, using the sequence of aptamer SEQ ID NO:5 as an origin,sequence optimization for strongest DNA polymerase binding can beperformed for every DNA polymerase known in the art. In this sense, thepresent disclosure also provides methods of screening for improvedaptamers for use in the disclosed methods.

Aptamers of the invention can contain any number of modifiednucleotides, internal and external linker and moieties and otherstructural modifications as long as these modifications do not interferewith the DNA polymerase deactivation and then activation processesduring DNA synthesis. For example, if desirable in a specific assay,they can incorporate phosphorothioate bonds at their termini to protectthe aptamers from the exonuclease hydrolysis (Skerra, A., 1992). Thehairpin-type noncircular aptamers can contain non-complementary 5′ or 3′nucleotide sequences. Preference should be given to those structuralmodifications that help to deactivate the DNA polymerase and do notaffect the uracil-DNA glycosylase activation reaction. Both loop andstem fragments can be modified in the hairpin-type aptamers. Althoughthe loop segments described in Yakimovich, O. Yu., et al. (2003),Jayasena, S. D. (1999), and U.S. Pat. No. 5,693,502 to Gold, L. andJayasena, S. D., contain art-recognized conserved sequence motifs, FIGS.3, 5, and Example 3 herein surprisingly show that removal of the5-methyl group from deoxythymidine (corresponding modification isdeoxyuridine) at certain exemplary positions (SEQ ID NOS:6-12 in FIG. 2and SEQ ID NOS:14-20 in FIG. 4) has little or no effect on the aptamerpolymerase-inactivation performance (FIGS. 3A and 5A, respectively).Perhaps the most surprising results were obtained in the DNApolymerase-activation assays for noncircular (FIG. 3B) and circularaptamers (FIG. 5B) wherein two pairs of circular (SEQ ID NOS:16 and 17)and noncircular aptamers (SEQ ID NOS:8 and 9) having deoxyuridines atthe same location within the loop sequence did not facilitatereactivation of Taq DNA polymerase in the presence of the uracil-DNAglycosylase. This unexpected phenomenon is discussed herein in Example3. According to additional aspects of the invention, identification ofthese two ‘glycosylase-insensitive’ deoxyuridine positions within theart-recognized highly conserved loop sequence 5′-TTCTTAGCGTTT-3′ (SEQ IDNO:23) has utility for design and functional optimization of bothcircular and noncircular hairpin-shaped aptamers. Although a singledeoxyuridine modification was used in the examples provided herein, morethan one nucleotide modification can be successfully applied within theloop sequence, including within the loop sequence 5′-TTCTTAGCGTTT-3′(SEQ ID NO:23), to optimize aptamer properties for DNA polymeraseinactivation and activation processes.

Methods of the invention can be performed at any reaction temperatureswherein a DNA polymerase and uracil-DNA glycosylase express suitableactivity. Specificity of DNA synthesis is usually increased at highertemperatures, and therefore preference should be given to thermostableenzymes. The upper possible level of the reaction temperature can beselected based on the DNA polymerase stability. In the cases when auracil-DNA glycosylase is not stable at the desired reactiontemperature, the DNA polymerase activation can be initiated at lowerintermediate temperature wherein the aptamer-modifying enzyme is stableand active and then raised to the desired reaction temperature. In someembodiments, a DNA polymerase is preferably first deactivated bycontacting (e.g., by combining or mixing) with an aptamer before otherreaction components of the DNA synthesis are added. Amount of an aptamerused in the reactions is an important factor. Molar reactionconcentration of an aptamer applied should be at least equal to theconcentration of a DNA polymerase or preferably greater. Marketproviders commonly do not disclose the molar amount of the enzymes,therefore the precise excess of the aptamers over the DNA polymerasesused in Examples provided herein was not known. However, it wasanticipated to be in a range of ˜40-80 fold, or even greater. In someembodiments, the aptamer is present in a molar excess (ratio) over theDNA polymerases of at least ˜10-50 fold, although the ratio can behigher or lower than 10-50 fold. In any case, as will be immediatelyunderstood by one of ordinary skill in the art, the amounts of theenzymes, aptamers and other reaction components used in the reaction maybe optimized and depend on many factors including, but not limited toselection of the particular enzymes, enzymatic activities at thereaction temperature, reaction temperature itself, nature of theaptamers, etc. Methods of the present invention can be particularlyuseful for so-called ‘fast’ PCR with a cycle time shorter than 20seconds. Rapid cycling requires use of elevated amounts of DNApolymerase and oligonucleotide primers increasing possibility ofnon-specific reactions.

In certain embodiments, methods of the invention can be practiced usinga kit comprising a DNA polymerase-binding oligonucleotide aptamer thatis capable of forming a hairpin structure wherein the loop sequencecomprises one or more deoxyuridine nucleotides recognizable andmodifiable by a uracil-DNA glycosylase enzymatic activity and anuracil-DNA glycosylase to provide for specific modification of theaptamer. The kit can also include a corresponding DNA polymerase whichneeds to be deactivated by the provided oligonucleotide aptamer.Alternatively, the kit can include, in addition to the aptamer-modifyingenzyme, a complex of the DNA polymerase with oligonucleotide aptamerwherein the components of this complex are present at a specific andoptimal molar ratio. As a matter of convenience, such kit can includeelements allowing the storage, transport and other reaction componentssuch as oligonucleotides, buffering components, additives, reactionenhancers, etc. The aptamers of the kits can be circular or noncircular,and they can incorporate more than one deoxyuridine within theirrespective loop segments. The kits can be used for DNA synthesis, DNAamplification as well as for detection and/or measurement/quantificationof amplified DNA fragments.

In some embodiments, the invention includes a reaction mixture for usein a method of DNA synthesis, which reaction mixture comprises: (i) aDNA polymerase, and (ii) a DNA polymerase-binding hairpin-likeoligonucleotide aptamer that incorporates one or more deoxyuridinenucleotide(s) within the loop sequence portion that are modifiable by auracil-DNA glycosylase enzymatic activity, and wherein the aptamer ispresent in an amount sufficient to inhibit DNA synthesis activity of theDNA polymerase in the reaction mixture, and other reaction componentsnecessary for DNA synthesis is also a subject of the present invention.The reaction mixtures may also include (iii) a uracil-DNA glycosylase.In some embodiments, the reaction mixture can be assembled usingconcentrated stock solutions of one or more components, usually in waterto provide the desired component concentration in the final mixture.Mixing is recommended to be performed at a low temperature (e.g., closeto 0° C.) at which the enzymes, particularly uracil-DNA glycosylases,are inactive. Preferably, the reaction mixture should be used for DNAsynthesis soon after preparation. Storage of a fully assembled reactionmixture is not recommended. However, reaction components includingenzymes can retain activity for long time (days, months, or even years)in a dried state. For example, in some embodiments of the invention, oneor more of the components for forming a mixture of the invention can beprovided in a dried form, such as dried beads as described, for example,in U.S. Pat. No. 3,721,725 to Briggs, A. R. and Maxwell, T. J. (1973),including (but are not limited to) DNA polymerase, oligonucleotideaptamer, and uracil-DNA glycosylase(s) such that one or more of thecomponents is prepared in a form of dried beads as described, forexample, in U.S. Pat. No. 3,721,725 to Briggs, A. R. and Maxwell, T. J.(1973). In some embodiments, the mixture comprises DNA polymerase, anoligonucleotide aptamer that binds to the DNA polymerase and present inan amount effective to inhibit DNA synthesis activity of the DNApolymerase, and a uracil-DNA glycosylase(s) that is capable of modifyingthe oligonucleotide aptamer to reduce or eliminate binding of theoligonucleotide aptamer to the DNA polymerase, which are mixed togetherin a dried form.

Example 1 Synthesis of Aptamers, Primers and Fluorescent Probes

Standard phosphoramidites, including modified nucleotide analogs such asdeoxyuridine (Catalog Number: 10-1050-xx), a phosphoramidite forincorporation of 5′-phosphate moiety, solid supports and reagents toperform the solid support oligonucleotide synthesis were purchased fromGlen Research. A 0.25 M 5-ethylthio-1H-tetrazole solution was used as acoupling agent. Oligonucleotides were synthesized either on ABI394 DNAsynthesizer (Applied Biosystems) or MerMaid 6 DNA synthesizer(BioAutomation Corporation) using protocols recommended by themanufacturers for 0.2 μmole synthesis scales. Fluorescein (FAM)conjugated to 5-position of deoxyribouridine (U) of probe SEQ ID NO:21(FIG. 6) was introduced to the hairpin during oligonucleotide synthesisusing 5′-dimethoxytrityloxy-5-[N-((3′,6′-dipivaloylfluoresceinyl)-aminohexyl)-3-acrylimido]-2′-deoxyribouridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research, Catalog Number: 10-1056-xx). A 6-fluorescein reportingdye was incorporated onto the 5′-end, and a BHQ1 quencher was introducedto the 3′-end of probe SEQ ID NO:3 (FIG. 1) using respectivephosphoramidite and CPG from Biosearch Technologies (Catalog numbers:BNS-5025 and BG1-5041G). After the automated synthesis, oligonucleotideswere deprotected in aqueous 30% ammonia solution by incubation for 12hours at 55° C. or 2 hours at 70° C.

Tri-ON oligonucleotides were purified by HPLC on a reverse phase C18column (LUNA 5 μm, 100 A, 250×4.6 mm, Phenomenex Inc.) using gradient ofacetonitrile in 0.1 M triethyl ammonium acetate (pH 8.0) or carbonate(pH 8.5) buffer with flow rate of 1 ml/min. A gradient profile includingwashing stage 0→14% (10 sec), 14→45% (23 min), 45→90% (10 min), 90→90%(5 min), 90→0% (30 sec), 0→0% (7 min) was applied for purification ofall Tri-ON oligonucleotides. The product containing fractions were drieddown in vacuum (SPD 1010 SpeedVac, TermoSavant) and trityl groups wereremoved by treatment in 80% aqueous acetic acid at room temperature for40-60 min. After addition to the detritylation reaction (100 μl) of 20μl sodium acetate (3 M), the oligonucleotide components wereprecipitated in alcohol (1.5 ml), centrifuged, washed with alcohol anddried down. Concentration of the oligonucleotide components wasdetermined based on the optical density at 260 nm and the extinctioncoefficients calculated for individual oligonucleotides using on-lineOligoAnalyzer 3.0 software provided by Integrated DNA Technologies.Based on the measurements, convenient stock solutions in water wereprepared and stored at −20° C. for further use. The purity of allprepared oligonucleotide components was confirmed by analytical 8-20%PAAG electrophoresis, reverse phase HPLC and by spectroscopy on Cary4000 UV-VIS spectrophotometer equipped with Cary WinUV software, BioPackage 3.0 (Varian, Inc.).

Example 2 Synthesis of Circular Oligonucleoside Aptamers

Exemplary circular stem-loop aptamers SEQ ID NOS:13-20 were prepared byligation of the corresponding 5′-phosphate incorporatingoligonucleotides shown in FIG. 4 using a T4 DNA Ligase kit from NewEngland Biolabs (Catalog number: M0202M). The reaction mixtures wereprepared by mixing 67 μl of 10× ligation buffer and 2.5 μl of T4 DNAligase (2,000 U/μl) from the kit with 10 optical units (at 260 nm) of a5′-phosphate-labelled oligonucleotide (FIG. 4) and deionized water toprovide 670 μl of the final reaction volume. The reaction mixtures wereleft at room temperature for 1 hour and then heated to 65° C. for 15min. According to HPLC analysis, the ligation reactions were nearlyquantitative (˜95%), and the circular aptamers were isolated by HPLCchromatography as described in Example 1. The collected fractions weredried down, and the circular aptamers were dissolved in water.Concentration of the circular aptamers was determined based on theoptical density at 260 nm as also described in Example 1. Based on themeasurements, convenient stock solutions in water were prepared andstored at −20° C. for further use.

Example 3 Application of Hairpin-Like Aptamers Containing DeoxyuridineNucleotide within the Loop Sequence to Control Polymerase Activity ofTaq Polymerase

This working example shows application of hairpin-like aptamerscontaining deoxyuridine nucleotide within the loop sequence to controlactivity of Taq polymerase during PCR.

For the results shown in FIGS. 3 and 5, reaction mixtures (25 μL) wereprepared on ice by mixing corresponding stock solutions to provide 200nM forward primer (FIG. 1, SEQ ID NO:1), 300 nM reverse primer (SEQ IDNO:2), 200 nM FRET probe (SEQ ID NO:3), 0.02 U/μL Taq DNA polymerase(GenScript cat no: E00007), dNTPs (200 μM each), bovine serum albumin(0.1 μg/μL), 100 ng of human genomic DNA (GenScript cat no: M00094) and,when present, one of the aptamers SEQ ID NOS:5-20 (80 nM) in 5 mM MgCl₂,50 mM KCl, 20 mM Tris-HCl (pH8.0). The reaction tubes were quicklytransferred into SmartCycler instrument (Cepheid Corporation) andtemperature cycling initiated. The PCR time/temperature profilecomprised initial incubation at 95° C. for 15 seconds followed by 50cycles of incubation at 95° C. for 1 second and then at 60° C. for 20seconds. The reaction fluorescence was measured in every PCR cycleduring the annealing/extension stage (60° C.) and the results are shownin FIGS. 3 and 5. Each fluorescence curve is an average of fouridentical reactions. Initial background fluorescence was subtracted bythe instrument software.

The reaction conditions used to generate the fluorescence profiles shownin FIGS. 3 and 5 were identical except for the presence or absence ofuracil-DNA glycosylase enzymatic activity and the presence or absence ofdifferent oligonucleotides as potential inhibitors of polymeraseactivity. PCR reactions were performed either in the absence (left panelof each figure) or in the presence (right panel of each figure) of AfuUracil-DNA Glycosylase (0.016 U/μL, New England Biolabs cat no: M0279S)in the reaction mixtures. Structures of the oligonucleotide aptamersused in the experiments of FIGS. 3 and 5 are shown in FIGS. 2 and 4,respectively.

In summary of this working Example, FIG. 3A shows that thedeoxyribouridine nucleotide modification within the hairpin loop (SEQ IDNOS:6-12) does not affect the ability of a noncircular aptamer todeactivate Taq DNA polymerase. All eight aptamers investigated(unmodified SEQ ID NO:5 and modified aptamers SEQ ID NOS:6-12) are veryeffective in blocking the DNA polymerase during PCR. No fluorescencesignal was detected when an aptamer was present in the reaction mixture.Addition of Afu Uracil-DNA Glycosylase effectively removes the DNApolymerase blockage, but not in all cases. As anticipated, unmodifiedaptamer SEQ ID NO:5 did not respond to the presence of the glycosylasein the reaction mixture. Surprisingly, three out of sevendeoxyuridine-modified aptamers, particularly aptamers SEQ ID NOS:8-10were refractory to DNA polymerase activation by uracil-DNA glycosylase(FIG. 3B), although to different degrees. Aptamers SEQ ID NOS:8 and 9effectively blocked the DNA polymerase regardless of the presence ofglycosylase. In the case of aptamer SEQ ID NO:10, theglycosylase-induced activation of the DNA polymerase was not complete.One possible explanation is that the uracil-DNA glycosylase cannotcleave the uracil base at those specific loop locations of aptamers SEQID NOS:8 and 9, and that there is a somewhat reduced rate of the uracilcleavage for aptamer SEQ ID NO:10. This hypothesis is supported by theart-recognized fact that loop sequences in hairpin-shapedoligonucleotides, especially short ones as in the aptamers exemplifiedherein, are structurally constrained, and this might explain by some ofthe nucleotides are inaccessible or partially accessible for recognitionby nucleases, glycosylases and other DNA-modifying enzymes. Theparticular loop sequence 5′TTCTTAGCGTTT3′ (SEQ ID NO:23) used in designof the hairpin aptamers herein is known in the art to be highlyconserved (e.g., Yakimovich, O. Yu., et al. (2003), Jayasena S. D.(1999), and U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D.).However, according to surprising aspects of the present invention, thissequence conservation does not exclude a possibility that loss of thesubstituted bases at the aptamer loop locations discussed hereinprovides for retention of the DNA polymerase-inactivating capabilitiesin the case of aptamers SEQ ID NOS:8 and 9 and partially in the case ofSEQ ID NOS:10.

The circular hairpin-shaped aptamers SEQ ID NOS:13-20 shown in FIG. 4were also investigated in PCR assays (FIGS. 5A and 5B) as ligands forreversible inactivation of Taq DNA polymerase. Regardless of shorterstem lengths (14 base pairs for SEQ ID NOS:13-20 vs. 17 base pairs forSEQ ID NOS:5-12) and highly elevated A-T base pair content, many ofthese circular aptamers effectively blocked Taq DNA polymerase duringPCR (FIG. 5A). Only three aptamers, in particular SEQ ID NOS:14, 19, and20 provided incomplete inactivation of the polymerase, where aptamer SEQID NO:19 provided the least inhibitory effect. Since unmodified circularaptamer SEQ ID NO:13 was very effective in polymerase blockage, the PCRresults obtained for aptamers SEQ ID NOS:14, 19, and 20 (FIG. 5A)indicate a likely positive hydrophobic contribution to aptamer-DNApolymerase complex formation, that is mediated by the 5-methyl group ofthymine, which is absent at the corresponding deoxyuridine-modifiedlocations within the loop sequence. The set of circular aptamers SEQ IDNOS:13-20 revealed glycosylase-induced polymerase reactivation patterns(FIG. 5B) similar to that observed for noncircular aptamers SEQ IDNOS:5-12 (FIG. 3B), with one exception. Unlike its counterpartnoncircular aptamer SEQ ID NO:10 (FIG. 3B), circular aptamer SEQ IDNO:18 effectively provided for reactivation of the inactivated DNApolymerase in the presence of uracil-DNA glycosylase.

Example 4 Kinetics of Activation by Uracil-DNA Glycosylase of VariousDNA Polymerases Initially Blocked by Deoxyuridine-Containing Aptamer

This working example shows the kinetics of activation by uracil-DNAglycosylase of Taq (GenScript cat no: E00007), Q5® (New England Biolabscat no: M0491S), Vent® (New England Biolabs cat no: M0254S), Deep Vent®(New England Biolabs cat no: M0258S), Bst large fragment (New EnglandBiolabs cat no: M0275S), and Phusion® (New England Biolabs cat no:M0530S) DNA polymerases initially blocked by a deoxyuridine-containingaptamers.

For FIG. 7, reaction mixtures (25 μL) were prepared on ice by mixingcorresponding stock solutions to provide self-priming hairpin SEQ IDNO:21 (200 nM, FIG. 6), a DNA polymerase (0.008 U/μL), dNTPs (200 μMeach), bovine serum albumin (0.1 μg/μL) and, when present, thenoncircular aptamer SEQ ID NO:12 (80 nM, FIG. 2) and Afu Uracil-DNAGlycosylase (0.016 U/μL, New England Biolabs cat no: M0279S) in 5 mMMgCl₂, 50 mM KCl, 20 mM Tris-HCl (pH8.0). During preparation of thereaction mixture, the self-priming hairpin (SEQ ID NO:21) and uracil-DNAglycosylase were always added last to a premixed solution. Then thereaction tubes were transferred into a SmartCycler instrument (CepheidCorporation) and heated to 60 or 65° C. as indicated for eachfluorescence profile in FIG. 7. The reaction fluorescence was monitoredevery 7 seconds. The plotted curves are the averages of four paralleledidentical reactions. Initial background fluorescence was subtracted.

The results of FIG. 7 show that not only Taq polymerase, but also manyother DNA polymerases can be inactivated and then activated usingaptamers of the present invention in the presence of uracil-DNAglycosylase. Only one of six investigated exemplary DNA polymerases,particularly Bst DNA polymerase, was not inactivated by noncircularaptamer SEQ ID NO:12. Other DNA polymerases showed an inactivation inthe presence of this aptamer as well as gradual uracil-DNAglycosylase-induced activation, although the efficiency of bothprocesses was variable among the individual DNA polymerases.

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The invention claimed is:
 1. A kit for activating an aptamer-inactivatedDNA polymerase, comprising: a uracil-DNA glycosylase enzymatic activity;and a DNA polymerase-binding oligonucleotide aptamer sequence that iscapable of forming a hairpin structure having a stem sequence portionand a loop sequence portion, wherein the loop sequence portion comprisesone or more deoxyuridine nucleotides modifiable by the uracil-DNAglycosylase enzymatic activity; and wherein the glycosylase modificationis sufficient to reduce or eliminate the binding of the oligonucleotideaptamer to the DNA polymerase and thereby provide for reactivating theDNA synthesis activity of the DNA polymerase.
 2. The kit of claim 1,wherein the loop sequence portion comprises a nucleotide sequence5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) wherein one or more thymidinenucleotides at positions 1, 2, 10, 11, and 12 of the SEQ ID NO:23sequence are substituted by one or more deoxyuridine nucleotides.
 3. Thekit of claim 1, wherein the oligonucleotide aptamer is a circularmolecule.
 4. The kit of claim 1, wherein the uracil-DNA glycosylaseenzymatic activity is effective to modify the oligonucleotide aptamer bygenerating at least one abasic site within the loop sequence portion. 5.The kit of claim 1, wherein the uracil-DNA glycosylase comprises AFUuracil-DNA glycosylase.
 6. The kit of claim 1, further comprising a DNApolymerase to provide a reaction mixture, wherein the DNApolymerase-binding oligonucleotide aptamer comprises a hairpin structurehaving a stem sequence portion and a loop sequence portion, wherein theloop sequence portion comprises one or more deoxyuridine nucleotidesmodifiable by the uracil-DNA glycosylase enzymatic activity, and whereinthe aptamer is present in an amount sufficient to inhibit DNA synthesisactivity of the DNA polymerase in the reaction mixture.
 7. The kit ofclaim 6, wherein the uracil-DNA glycosylase enzymatic activity issufficient, under suitable conditions, to modify the oligonucleotideaptamer to reduce or eliminate binding of the oligonucleotide aptamer tothe DNA polymerase, thereby activating or enhancing the DNA synthesisactivity of the DNA polymerase.
 8. The kit of claim 7, wherein at leastone of the DNA polymerase activity and/or the uracil-DNA glycosylaseenzymatic activity is temperature dependent.
 9. The kit of claim 8,wherein the uracil-DNA glycosylase enzymatic activity increases withincreasing temperature of the reaction mixture from a first temperatureto a second temperature that activates or more strongly facilitates theuracil-DNA glycosylase enzymatic activity.
 10. The kit of claim 6,wherein the DNA polymerase, and/or oligonucleotide aptamer, and/oruracil-DNA glycosylase enzymatic activity are present in the reactionmixture in a dried state.
 11. The kit of claim 6, wherein the loopsequence portion comprises a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQID NO:23) wherein one or more thymidine nucleotides at positions 1, 2,10, LI, and 12 of the SEQ ID NO:23 sequence are substituted by one ormore deoxyuridine nucleotides.
 12. The kit of claim 6, wherein theoligonucleotide aptamer is a circular molecule.
 13. The kit of claim 7,wherein the uracil-DNA glycosylase enzymatic activity is effective tomodify the oligonucleotide aptamer by generating at least one abasicsite within the loop sequence portion.
 14. The kit of claim 7, whereinthe uracil-DNA glycosylase comprises AFU uracil-DNA glycosylase.
 15. Thekit of claim 6, wherein the mixture further comprises one or more ofdATP, dCTP, dGTP, and/or dTTP, and/or Mg²⁺ ion.